Two processes, the binding of appropriate ligands to membrane-bound receptors and the receptor-controlled flux rates of specific inorganic ions, are important in determining the transmission of signals between nerve cells, and constitute some of the limits within which the nervous system can function. Understanding these two processes and their relationship is the objective of our studies. Investigations of the relationship between the ligand binding process and the receptor controlled flux rates under well defined conditions have become possible with membrane vesicles containing acetylcholine receptor from the electric organ of E. electricus. The proposed investigations are based on methods and techniques which we have developed in the 1st four years. (1) The separation of vesicles containing functional receptors from those which do not, so that we can investigate controlled fluxes without interference from other processes. (2) The adaptation of flow quench techniques so that one can measure flux rates in the msec to min time region. (3) The application of fluorescence correlation spectroscopy so that we can determine both the size of, and number of receptor sites per, vesicle. With a 3-stage flow quench apparatus we are able to measure 4 individual steps involved in regulating the receptor-controlled flux: (1) Receptor-controlled influx of ions; (2) the rate of ligand-induced inactivation of the receptor; (3) the rate of flux mediated by equilibrium mixtures of active and inactive forms of the receptor, and (4) the rate of receptor reactivation. In addition to comparing the effect of different ligands and interesting inhibitors on the individual steps, we plan to investigate additional factors which may affect receptor controlled flux: (1) the concentration of inorganic ions in the vesicles; (2) transmembrane voltage, and (3) Ca2 ion. We have already succeeded in predicting receptor controlled flux over a 200-fold concentration range of carbamylcholine. We are working on the theory which relates the flux measurements to time-dependent changes in transmembrane potential. We are developing the techniques and making the measurements with cells which will allow us to compare theory with experiment.